Efrapeptin and adenyllylimidodiphosphate (AMPPNP) block the access of added ligands to the catalytic site of F1, the terminal coupling factor of mitochondrial oxidative phosphorylation. Using efrapeptin and AMPPNP0as protecting agents, the catalytic site of F1 will be modified by arginine- and lysine-specific reagents and by photoaffinity analogs of ATP. The labeled subunits will be examined by gel electrophoresis, peptide mapping, amino acid analysis, and sequencing to characterize the catalytic center. Based on differential incorporation of 8-azido-ATP and 8-azido-AMPPNP by F1 and nucleotide-depleted F1, it may be possible to distnguish between regulatory and catalytic adenine nucleotide binding sites on the enzyme and to determine the stochiometry and subunit location of these sites. The results may also provide further information regarding cooperative interactions between catalytic subunits and the number and type(s) of catalytic sites on the enzyme. Further studies of the mechanism of regulation of F1 by the native inhibitor protein will include study of factors which affect the binding of the inhibitor to the enzyme. We will test the possibilities that the inhibitor protein masks the catalytic site of F1 and that the inhibitor is reversibly phosphorylated as a required step in its mode of action.